Biofilm formation of Streptococcus mutans, Streptococcus sanguinis, Staphylococcus epidermidis, Staphylococcus aureus, Lactobacillus casei, and Candida Albicans on 5 thermoform and 3D printed orthodontic clear aligner and retainer materials at 3 time points: an in vitro study.

INTRODUCTION: Orthodontic clear aligners and retainers have numerous advantages that is making them ever increasingly popular. However, they might, similar to any other oral appliance, contribute to biofilm formation and finally dental caries or white spot lesions or gingival inflammations. The literature on biofilm formation on orthodontic clear appliances is very scarce and limited to a few microorganisms and materials. Therefore, this experimental study evaluated the biofilm formation on 5 thermoformed and 3D printed CAD/CAM orthodontic retainers in 3 intervals. METHODS: In this in vitro study, 345 specimens (270 test discs and 45 negative controls) were created from fabricated retainers. Retainers included a 3D printed CAD/CAM material (Detax) and four thermoformed retainers [Erkodent (polyethylene terephthalate glycol [PETG]); EasyVac (polyethylene); DB (polyester based on terephthalic acid); and Clear Tech]. They were all 1 mm thick, and all completely fabricated, i.e., heated or printed. The discs were placed in 96-well plates. Microorganisms were cultured on 270 discs for 24 h (90 discs), 72 h (90 other discs), and 5 days or 120 h (90 other discs). Biofilm formation of the strains and negative controls was measured using the microtiter plate assay by ELISA reading. The microbes' ability to produce biofilm was categorized based on the comparison of average optical density (OD) of tests versus a cut-off point OD (ODc) calculated as the average of the OD of corresponding negative controls plus 3× its standard deviation: non-biofilm former [OD ≤ ODc], weak biofilm former [ODc < OD ≤ (2 × ODc)], moderate biofilm former [(2 × ODc) < OD ≤ (4 × ODc)], and strong biofilm former [(4 × ODc) < OD]. These were also converted to ranked scores between zero (no biofilm) and 3. The difference between ODs with control ODs were calculated. These were analyzed using 3-way ANOVA, 2-way ANOVA, and Tukey tests (α = 0.05, α = 0.008). RESULTS: The 3-way ANOVA showed that the overall difference among the ΔODs of 5 retainers (all microorganisms and all intervals combined, n = 270) was not significant (F = 1.860, P = 0.119). Nevertheless, the difference among 3 intervals (F = 31.607, P = 0.0000) and the difference among the 6 microorganisms (F = 24.044, P = 0.0000) were significant. According to the Tukey test, the differences between the 1st interval with either of the other two intervals was significant (both P values = 0.000). There were significant differences between Candida albicans with all other organisms (all 5 P values = 0.0000). All other pairwise comparisons were insignificant (all 10 P values ≥ 0.1). After taking the averages of the 3 intervals, the order of the biofilm generation for different materials were as follows: Detax (average score: 1.56), Easyvac (1.67), Erkodent (1.78), Clear Tech (1.83), BD (2.28). CONCLUSIONS: As far as these 6 microorganisms are of concern, there might not be a significant overall difference among the clear retainer materials tested in this study. A significant overall increase was observed between the first and third days, which later did not significantly increase more until day 5. The Candida albicans biofilm was more intense than the tested 5 bacteria, which themselves showed rather similar growth patterns to each other.

Streptococcus mutans counts in patients wearing removable retainers with silver nanoparticles vs those wearing conventional retainers: A randomized clinical trial.

INTRODUCTION: The rough surface of Hawley removable appliances provides an environment for plaque accumulation, leading to enamel demineralization. The aim of this study was to evaluate the effect of silver nanoparticles incorporated into acrylic baseplates of orthodontic retainers on Streptococcus mutans colony-forming units. METHODS: Sixty-six orthodontic patients at the debonding stage were randomly assigned to 2 sex-matched groups with stratified block randomization: group 1 received conventional removable retainers; group 2 received removable retainers containing silver nanoparticles (about 40 nm in size and 500 ppm in concentration). After comprehensive orthodontic treatment, patients who revealed no clinical evidence of dental caries, periodontal pockets, or systemic disease were considered eligible for this study. Swab samples were taken from the maxillary palatal side from the patient in the dental chair at retainer placement (T1, 1 week after debonding the fixed orthodontic appliance) and T2, 7 weeks later. The main outcome was to compare the number of S mutans colony-forming units between the 2 groups 7 weeks after retainer delivery. The results were analyzed by using analysis of covariance. The participants and the assessors were blinded to the allocation groups. RESULTS: Twenty-nine patients in the control group and 32 in the intervention group were analyzed. At T1, the intervention group had higher S mutans colony counts relative to the control group. The analysis of covariance test showed a significant reduction of colonies in the intervention group after 7 weeks. The mean difference of colony counts between the 2 groups was 40.31 (95% confidence interval, 24.83-55.79; P < 0.001). CONCLUSIONS: Adding silver nanoparticles to the acrylic plate of retainers had a strong antimicrobial effect against S mutans under clinical conditions. REGISTRATION: This study was registered as a clinical trial at the Iranian Clinical Trial Center under the code number IRCT201309239086N2. FUNDING: This trial was supported by Hamadan Dental Research Centre, Hamadan University of Medical Sciences, Hamadan, Iran.

Quantitative analysis of changes in salivary mutans streptococci after orthodontic treatment.

INTRODUCTION: The purpose of this study was to analyze the initial changes in salivary mutans streptococci levels after orthodontic treatment with fixed appliances. METHODS: Our subjects consisted of 58 adults. Whole saliva and simplified oral hygiene index values were obtained at 4 time points: at debonding (T1), 1 week after debonding (T2), 5 weeks after debonding (T3), and 13 weeks after debonding (T4). Repeated measures analysis of variance was used to determine the time-related differences in salivary bacterial levels and the simplified oral hygiene index values among the 4 time points after quantifying the salivary levels of Streptococcus mutans, Streptococcus sobrinus, and total bacteria with real-time polymerase chain reaction. RESULTS: Simplified oral hygiene index values and total bacteria significantly decreased, but salivary mutans streptococci levels significantly increased after orthodontic treatment. The amounts of total bacteria in saliva significantly decreased at T3 (T1, T2 > T3, T4), and the simplified oral hygiene index values decreased at T2 (T1 > T2, T3, T4). However, salivary S mutans and S sobrinus significantly increased at T3 and T4, respectively (T1, T2 < T3 < T4). Furthermore, the proportion of mutans streptococci to total bacteria significantly increased at T4 (T1, T2, T3 < T4). CONCLUSIONS: This study suggests that careful hygienic procedures are needed to reduce the risk for dental caries after orthodontic treatment, despite overall improved oral hygiene status.

An assessment of the effectiveness of mechanical and chemical cleaning of Essix orthodontic retainer.

OBJECTIVES: To determine the effectiveness of mechanical and chemical cleaning on the removal of microorganisms from Essix orthodontic retainers. DESIGN: In vitro laboratory study. SETTING: Department of Orthodontics and Microbiology, Eastman Dental Institute, University College London, UK. METHODS: Study 1: 120 Essix retainers were divided into four cleaning groups. The effectiveness of each cleaning method to remove a single species biofilm of Streptococcus mutans from the retainer was assessed. Study 2: 140 Essix retainers were divided into four study groups (brushing with fluoride toothpaste, chlorhexidine gel, immersion in chlorhexidine solution only and a control) to investigate the chemical and mechanical cleaning of the multispecies biolfilm of (Streptococcus sanguis, Actinomyces naeslundii, methicillin-resistant Staphylococcus aureus and Candida albicans). RELEVANT RESULTS: In study 1, brushing with toothpaste resulted in 99% reduction of Streptococcus mutans. In study 2, all three cleaning methods recorded similarly statistically significant reductions in colony forming units per millilitre compared to the control. There were no statistically significant differences between any of the cleaning groups for any of the microorganisms except MRSA-16. For MRSA-16, chlorhexidine mouthwash and gel were significantly more potent in eliminating the microorganism than the fluoride toothpaste. CONCLUSIONS: All three cleaning methods effectively removed 99% of microorganisms from the Essix retainers. Brushing with fluoride toothpaste can therefore be confirmed as an effective method for cleaning retainers in most circumstances. The use of chlorhexidine gel or mouthwash is recommended in patients where bacterial infection has to be avoided due to immunosuppression or other reasons.

Biofilm formation on stainless steel and gold wires for bonded retainers in vitro and in vivo and their susceptibility to oral antimicrobials.

OBJECTIVE: Bonded retainers are used in orthodontics to maintain treatment result. Retention wires are prone to biofilm formation and cause gingival recession, bleeding on probing and increased pocket depths near bonded retainers. In this study, we compare in vitro and in vivo biofilm formation on different wires used for bonded retainers and the susceptibility of in vitro biofilms to oral antimicrobials. MATERIALS AND METHODS: Orthodontic wires were exposed to saliva, and in vitro biofilm formation was evaluated using plate counting and live/dead staining, together with effects of exposure to toothpaste slurry alone or followed by antimicrobial mouthrinse application. Wires were also placed intra-orally for 72 h in human volunteers and undisturbed biofilm formation was compared by plate counting and live/dead staining, as well as by denaturing gradient gel electrophoresis for compositional differences in biofilms. RESULTS: Single-strand wires attracted only slightly less biofilm in vitro than multi-strand wires. Biofilms on stainless steel single-strand wires however, were much more susceptible to antimicrobials from toothpaste slurries and mouthrinses than on single-strand gold wires and biofilms on multi-strand wires. Also, in vivo significantly less biofilm was found on single-strand than on multi-strand wires. Microbial composition of biofilms was more dependent on the volunteer involved than on wire type. CONCLUSIONS: Biofilms on single-strand stainless steel wires attract less biofilm in vitro and are more susceptible to antimicrobials than on multi-strand wires. Also in vivo, single-strand wires attract less biofilm than multi-strand ones. CLINICAL SIGNIFICANCE: Use of single-strand wires is preferred over multi-strand wires, not because they attract less biofilm, but because biofilms on single-strand wires are not protected against antimicrobials as in crevices and niches as on multi-strand wires.

Preclinical in vitro study of streptococcus mutans accumulation in three fixed retainer designs: Microbiological assay.

The use of fixed retainers in the lower arch is frequent; however, its presence increases the accumulation of biofilm and dental calculus. The objective of this research was to evaluate, in vitro, the accumulation of Streptococcus mutans (S. mutans) in 3 designs of fixed retainers. Nine models were reproduced in heat-cured acrylic resin and divided into groups: straight retainer (SR), retainer with vertical strap (RVS), retainer with horizontal strap (RHS). The accumulation of S. mutans was assessed using the MTT assay (3-4,5-dimethyl-thiazol-2-yl-2,5-diphenyltetrazolium bromide) and then measured using an automated reader. The RHS group showed less biofilm accumulation compared to the other groups (p<0.05). The distance between the tooth surface and the retainer showed a strong negative correlation with biofilm accumulation (rs=-0.79, p=0.00037). The RHS showed significantly less accumulation of S. mutans due to the distance between the retainer and the tooth surface. This research provides relevant data for a future randomized clinical trial.

Influence of thermoplastic retainers on Streptococcus mutans and Lactobacillus adhesion.

INTRODUCTION: This study was designed to test the hypothesis that thermoplastic retainers influence oral microbial flora during the retention period because they prevent the flushing effect of saliva on dental and mucous tissues. METHODS: Twenty-four orthodontic patients finished the study. After debonding, the patients were given thermoplastic retainers (Essix ACE 0.040-in plastic, Dentsply International, York, Pa) for both jaws and instructed to wear them all day. Plaque samples from tooth surfaces and saliva samples were collected from each patient just after debonding (T0), and on day 15 (T1), day 30 (T2), and day 60 (T3) of retention. The jaws were divided into 6 regions, and the data for each region were evaluated separately. Total viable Lactobacillus and Streptococcus mutans colonies were counted, and the numbers of the viable microorganisms were calculated. RESULTS: The numbers of Lactobacillus colonies at T3 were higher than at T0, T1, and T2, and the difference between T0 and T3 was statistically significant (P <0.05). The numbers of S mutans colonies at T3 were higher than at T0, T1, and T2, and the differences between T0 and T1, and T1 and T2 were statistically significant (P <0.05). CONCLUSIONS: Retention with thermoplastic retainers might create oral conditions conducive to S mutans and Lactobacillus colonization on dental surfaces.

The prevalence of opportunistic pathogens associated with intraoral implants.

AIMS: The aim of this study was to determine the prevalence and proportions of opportunistic pathogens harboured on orthodontic retainers. METHODS AND RESULTS: First, Staphylococcus spp. and Candida spp. were isolated from the retainer's inner surface and from other mucosal surfaces of the subject's mouth by routine bacterial culture. The prevalence and proportions of these micro-organisms on retainers was compared in different areas of the mouth within a group of retainer wearers, and mucosal carriage was compared to a group of nonretainer wearers. Staphylococcus spp. were isolated from 50% of the retainers and comprised on average 8·4% of the viable microbiota. Candida spp. comprised 0·13% of the viable microbiota and were recovered from 66·7% of the retainers. Neither genus was isolated from nonretainer wearers. Second, the two most commonly worn retainers manufactured from different materials were sampled; again Staphylococcus spp. and Candida spp. were recovered; however, no statistical differences were observed between the devices. CONCLUSIONS: Opportunistic, nonoral, pathogenic micro-organisms were recovered from orthodontic retainers. SIGNIFICANCE AND IMPACT OF THE STUDY: It is possible that an orthodontic retainer could be a reservoir for opportunistic pathogens and act as a source of cross-, self- and re-infection.

Antibacterial activity and debonding force of different lingual retainers bonded with conventional composite and nanoparticle containing composite: An in vitro study.

BACKGROUND: To evaluate the antibacterial activity and debonding force of retainers bonded with conventional and nanoparticle (TiO2) containing composite. METHODOLOGY: Antibacterial activity was tested against Streptococcus mutans and Lactobacillus acidophilus using disk agar diffusion, biofilm inhibition, and eluted components tests. For the eluted components test, colony counts of bacteria were tested on 0, 3, and 30 days. Three different retainers were bonded to the lingual surface of extracted lower incisors using conventional and 1% TiO2 composite. Samples were divided as follows: Group 1: 1a, stainless steel retainer (Bond-a-Braid) with conventional composite, and 1b, stainless steel retainer with nanoparticle composite; Group 2: 2a, titanium retainer with conventional composite, and 2b, titanium retainer with nanoparticle composite; Group 3: 3a, fiber-reinforced retainer (Interlig) with conventional composite, and 3b, fiber-reinforced retainer with nanoparticle composite. The Instron stereomicroscope was used to test debonding force and failure sites respectively. RESULTS: In the disk agar diffusion test, TiO2 composite has shown more inhibition zones. Biofilm inhibition test showed a significant decrease in colony counts of both organisms in the TiO2 group. The eluted component test showed a significant decrease in colony counts from day 0 to day 30 in the TiO2 group compared with the control group. The highest debonding force was observed in stainless steel retainers with conventional composite, and lowest in fiber-reinforced composite retainers with TiO2 composite, with no significant difference in Adhesive Remnant Index scores. CONCLUSION: The TiO2 composite group showed greater antibacterial activity without compromising the bond strength, which was statistically significant. Compared with other groups, stainless steel wires bonded with conventional composite showed the highest debonding force.

Microbial evaluation of the effectiveness of different methods for cleansing clear orthodontic retainers: A randomized clinical trial.

OBJECTIVE: To compare effectiveness of brushing an Essix retainer with water only and brushing followed by soaking in one of three commercial cleansing tablets. MATERIALS AND METHODS: A double-blind, parallel, randomized clinical trial with a split-mouth design was completed with 60 volunteers with specific eligibility criteria assigned to three groups to compare the cleansing effectiveness of brushing an Essix retainer with water only, followed by soaking in one of three alkaline peroxide-based commercial cleansing tablets (Retainer Brite®, Kukis®, and Corega®). Each participant wore an upper Essix retainer (with an electronic microsensor) on a full-time basis for 14 days and used one of the three products on one side of the retainer for 15 min/d. The effectiveness of the cleansing tablets was tested by the scanning electron microscopy, bacterial identification, bacterial quantification, and disk diffusion methods. RESULTS: Bacterial quantification tests demonstrated nonsignificant statistical differences between the control and test sides of the three cleansing tablets: Retainer Brite®, Kukis®, and Corega® (Mann-Whitney test P-values were .6, .37, and .5, respectively). A Kruskal-Wallis test also showed nonstatistical difference in the bacterial counts between the three groups (P-value = .5). In vitro tests showed a minimal inhibition zone of Staphylococcus epidermidis only with Corega® tablets. CONCLUSIONS: Using chemical cleansing tablets after mechanical cleansing did not significantly reduce the bacterial count in Essix retainers when compared to use of mechanical cleansing alone. However, the tablets seem to be effective against "cocci" bacterial species.

Antimicrobial activity of a quaternary ammonium methacryloxy silicate-containing acrylic resin: a randomised clinical trial.

Quaternary ammonium methacryloxy silicate (QAMS)-containing acrylic resin demonstrated contact-killing antimicrobial ability in vitro after three months of water storage. The objective of the present double-blind randomised clinical trial was to determine the in vivo antimicrobial efficacy of QAMS-containing orthodontic acrylic by using custom-made removable retainers that were worn intraorally by 32 human subjects to create 48-hour multi-species plaque biofilms, using a split-mouth study design. Two control QAMS-free acrylic disks were inserted into the wells on one side of an orthodontic retainer, and two experimental QAMS-containing acrylic disks were inserted into the wells on the other side of the same retainer. After 48 hours, the disks were retrieved and examined for microbial vitality using confocal laser scanning microscopy. No harm to the oral mucosa or systemic health occurred. In the absence of carry-across effect and allocation bias (disks inserted in the left or right side of retainer), significant difference was identified between the percentage kill in the biovolume of QAMS-free control disks (3.73 ± 2.11%) and QAMS-containing experimental disks (33.94 ± 23.88%) retrieved from the subjects (P ≤ 0.001). The results validated that the QAMS-containing acrylic exhibits favourable antimicrobial activity against plaque biofilms in vivo. The QAMS-containing acrylic may also be used for fabricating removable acrylic dentures.

The influence of surface roughness and surface dynamics on the attachment of Methicillin-Resistant Staphylococcus aureus onto orthodontic retainer materials.

Staphylococci species have been isolated from removable orthodontic retainers. The aims of this study were to determine the most suitable device to analyze surface roughness of autopolymerized acrylic and thermoplastic materials and whether the surface dynamics of these materials influences the attachment of Methicillin-Resistant Staphylococcus aureus (MRSA). Clinically simulated samples of autopolymerized acrylic and thermoplastic material were first evaluated using laser non-contact, stylus mechanical profilometries and atomic force microscopy (AFM) followed by contact angle measurement to characterize their surface dynamics. Finally, an in vitro biofilm assay was carried out using a constant depth film fermentor to assess biofilm attachment. The results showed a significant difference between the roughness values obtained from the tested profilometers with the AFM exhibiting the most consistent roughness values. MRSA tended to accumulate initially within the microscopic irregularities of autopolymerized acrylic samples whereas acid-base and electron donor interactions influenced the bacterial attachment onto the thermoplastic samples.

Cleaning removable orthodontic appliances: a survey.

OBJECTIVE: The aim of this survey was to analyze the recommendations commonly followed in Germany for cleaning removable orthodontic appliances (ROAs) and to compare these with published procedures. METHODS: A total of 450 randomly-selected orthodontists were contacted by telephone and asked for their recommendations for cleaning ROA. RESULTS: The most frequent recommendation was mechanical cleaning with a toothbrush (99.8%). Chemical detergents (37.1%) and treatment with diluted vinegar or citric acid (30.5%) were mentioned less often. CONCLUSION: The use of cleaning solutions was recommended comparatively infrequently, even though the scientific literature suggests that this is the most hygienic way to care for ROA.